PU.1antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes

Abstract

Antisense long non-coding RNAs (AS lncRNAs) play important roles in refined regulation of animal gene expression. However, their functions and molecular mechanisms for domestic animal adipogenesis are largely unknown. Here, we found a novel AS lncRNA transcribed from the porcine PU.1 gene (also known as SPI1) by strand-specific RT-PCR. Results showed that PU.1 AS lncRNA was expressed and generally lower than the level of PU.1 mRNA in porcine subcutaneous adipose, heart, liver, spleen, lympha, skeletal muscle and kidney tissues. We further found that the levels of PU.1 mRNA and PU.1 protein were significantly lower in subcutaneous and intermuscular adipose than in mesenteric and greater omentum adipose, whereas the levels of PU.1 AS lncRNA showed no difference in porcine adipose tissues from four different parts of the body. During porcine adipogenesis, levels of PU.1 mRNA increased at day 2 and then gradually decreased. Meanwhile, PU.1 AS lncRNA exhibited an expression trend similar to PU.1 mRNA but sharply decreased after day 2. Interestingly, PU.1 protein level rose during differentiation. In addition, at day 6 after differentiation, knockdown of endogenous PU.1 promoted adipogenesis, whereas knockdown of endogenous PU.1 AS lncRNA had the opposite effect. Moreover, peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid synthase (FASN) were significantly upregulated in the PU.1 shRNA treatment group (P < 0.05), whereas they were downregulated in the PU.1 AS shRNA treatment group (P < 0.05). Adipose triglyceride lipase [ATGL; also known as patatin-like phospholipase domain containing 2 (PNPLA2)] and hormone-sensitive lipase [HSL; also known as lipase, hormone-sensitive (LIPE)] contrasted with PPARG and FASN. Finally, the PU.1 mRNA/PU.1 AS lncRNA duplex was detected by an endogenous ribonuclease protection assay combined with RT-PCR. Based on the above results, we suggest that PU.1 AS lncRNA (vs. its mRNA translation) promotes adipogenesis through the formation of a sense-antisense RNA duplex with PU.1 mRNA.

Long Noncoding RNAs in Metabolic Syndrome Related Disorders

“…Formation of this sense-antisense RNA duplex was also confirmed in porcine adipocytes by an endogenous ribonuclease protection assay combined with RT-PCR. In line with the previous data, expression of PPAR γ and fatty acid synthase (FASN) was significantly upregulated in the lncRNA PU.1 shRNA treated group [63]. …”

Long noncoding RNAs in regulating adipogenesis: new RNAs shed lights on obesity

“…Recent studies indicated that PU.1 AS lncRNA regulated adipogenesis through preventing PU.1 mRNA translation by forming an mRNA/AS lncRNA duplex [42, 43]. PU.1 is a transcription factor with inhibitory effects on preadipocyte differentiation [44].…”

Comparative analyses of long non-coding RNA in lean and obese pigs

“…At the time of publication, a total of 12,103 pig lncRNAs, 8,923 chicken lncRNAs and 8,250 cow lncRNAs are included in the ALDB database [16]. While analyzing lncRNA expression in pig, Zhao et al developed a systematic protocol for the identification and characterization of lncRNAs in fetal porcine skeletal muscle [17]. Moreover, an antisense lncRNA of the PU.1 gene was identified, which can form a sense-antisense RNA duplex to promote adipogenesis [18].…”